Recent investigations have established the feasibility of using exogenously delivered angiogenic growth factors to increase collateral artery development in animal models of myocardial and hindlimb ischemia. Objective: Our aim was to evaluate the ability of a stabilized form of acidic fibroblast growth factor (aFGF-S-117) to stimulate collateralization and arteriogenesis in the rabbit hindlimb following the surgical induction of ischemia by femoral artery extraction. A secondary objective was to examine angiogenic and arteriogenic effects of aFGF-S-117 in the absence of a peripheral blood flow deficit. Methods and results: Five days after femoral artery removal, aFGF-S-117 (1, 3, or 30 mug/kg) was intramuscularly delivered into the hindlimb, three times per week for 2 consecutive weeks. End-point measurements performed on day 20 found that hindlimb reserve blood flow was significantly improved in rabbits that received 3 or 30 mug/kg of aFGF-S-117. with no difference in efficacy between these two doses. These hemodynamic results were supported by angiographic evidence showing enhanced density of collateral vessels in the medial thigh region and histological findings of increased capillary density within the gastrocnemius muscle from rabbits treated with aFGF-S-117. When an efficacious dose of 3 mug/kg of aFGF was administered to sham-operated rabbits with intact femoral arteries, there was no change in any of the blood flow, angiographic or histological parameters measured. Conclusions: These findings demonstrate that a stabilized form of aFGF stimulated the development of functional collateral arteries in the rabbit hindlimb, an effect which was dependent upon removal of the femoral artery. These results suggest that aFGF- S-117 may have therapeutic potential for the treatment of arterial occlusive disorders. (C) 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Objective: Animal models of hindlimb ischemia are critical to our understanding of peripheral vascular disease and allow us to evaluate therapeutic strategies aimed to improve peripheral collateral circulation. To further elucidate the processes involved in revascularization following ischemia, we evaluated the temporal association between tissue ischemia, vascular endothelial cell growth factor (VEGF) release, angiogenesis (capillary sprouting), arteriogenesis (growth of the larger muscular arteries), and reserve blood flow (functional collateral flow). Methods: New Zealand White rabbits (male 3-4 kg) were evaluated at specific days (0, 5, 10, 20 or 40) following femoral artery removal for measurement of hindlimb blood Row, skeletal muscle lactate production and VEGF content, capillary density (a marker of angiogenesis), and angiographic score (a marker of arteriogenesis). Results: Maximal capillary sprouting occurred within 5 days of femoral artery removal and was temporally associated with reduced resting hindlimb blood flow, increased lactate release and detectable levels of skeletal muscle VEGF. The growth of larger angiographically visible collateral vessels occurred after 10 days and was not temporally associated with ischemia or skeletal muscle VEGF content, but did coincide with a large functional improvement in the reserve blood flow capacity of the limb. Conclusions: Following femoral artery removal in the rabbit, the time course of angiogenesis and arteriogenesis were clearly distinct. Tissue ischemia and/or VEGF may stimulate capillary sprouting, but this response does not translate to a significant improvement in collateral flow. The growth and development of the larger collateral vessels was correlated with a large functional improvement in collateral Row, and occurred at a time when VEGF levels were undetectable. (C) 2001 Elsevier Science B.V. All rights reserved.

rRNA synthesis by RNA polymerase I requires both the promoter selectivity factor 1, which is composed of TATA binding protein (TBP) and three TBP-associated factors, and the activator upstream binding factor (UBF). Whereas there is strong evidence implicating a role for phosphorylation of UBF in the control of growth-induced increases in rRNA transcription, the mechanism of this effect is not known. Results of immunoprecipitation studies with TBP antibodies showed increased recovery of phosphorylated UBF from growth-stimulated smooth muscle cells. Moreover, using an immobilized protein-binding assay, we found that phosphorylation of UBF in vivo in response to stimulation with different growth factors or in vitro with smooth muscle cell nuclear extract increased its binding to TBP. Finally, we demonstrated that UBF-TBP binding depended on the C-terminal 'acidic tail' of UBF that was hyperphosphorylated at multiple serine sites after growth factor stimulation. Results of these studies suggest that phosphorylation of UBF and subsequent binding to TBP represent a key regulatory step in control of growth-induced increases in rRNA synthesis.

To identify cis- and trans-acting factors that regulate smooth muscle-specific gene expression, we studied the smooth muscle myosin heavy chain gene, a rigorous marker of differentiated smooth muscle. A comparison of smooth muscle myosin heavy chain promoter sequences from multiple species revealed the presence of a highly conserved 227-base pair domain (nucleotides -1321 to -1095 in rat). Results of a deletion analysis of a 4.3-kilobase pair segment of the rat promoter (nucleotides -4220 to +88) demonstrated that this domain was necessary for maximal transcriptional activity in smooth muscle cells. Gel-shift analysis and site-directed mutagenesis demonstrated that one true CArG and another CArG-like element contained within this domain were both recognized by the serum response factor and were both required for the positive activity attributable to this domain. Additional studies demonstrated that mutation of a GC-rich sequence within the 227-base pair conserved domain resulted in a nearly 100% increase in transcriptional activity. Gel-shift analysis showed that this GC-rich repressor element was recognized by both Sp1 and Sp3. These data demonstrate that transcriptional control of the smooth muscle myosin heavy chain gene is highly complex, involving both negative and positive regulatory elements, including CArG sequences found in the promoters of multiple smooth muscle differentiation marker genes.

Hypertrophy of vascular smooth muscle cells (VSMC) is an important adaptive response of hypertension. Drug intervention studies have implicated a role for angiotensin II (A-II) in the mediation of VSMC hypertrophy in vivo, and A-II is a potent hypertrophic agent for VSMC in culture. Our laboratory has previously shown that A-II-induced hypertrophy of cultured VSMC is due in part to generalized increases in protein synthesis and increased content of rRNA. The aim of the present study was to determine if A-II stimulates rRNA gene synthesis and whether the rRNA transcription factor, upstream binding factor (UBF), is involved. Nuclear run-on analysis demonstrated that A-II induced a greater than 5-fold increase in rRNA gene synthesis within 6 h of stimulation. A-II also stimulated a rapid increase in UBF phosphorylation as well as nucleolar localization, but no changes in the content of UBF. Phosphoamino acid analysis showed that phosphorylation occurred only on serine residue(s). Results demonstrate that increased transcription of ribosomal DNA contributes to the A-II-induced increase in protein synthesis and VSMC hypertrophy, and suggest that an important regulatory event in this pathway may be the phosphorylation and/or nucleolar localization of UBF.

We have previously shown a direct relationship (r = .97) between the fall in arterial blood pressure and the increase in skeletal muscle oxygen extraction (MVO2) during canine endotoxemia. Since it is well known that hypotension activates the sympathetic system, the primary aim of these experiments was to determine if the increase in MVO2 during endotoxemia is a result of elevated levels of catecholamines due to increased sympathetic neural and/or humoral activity (sympathoadrenal system). Canine gracilis muscles were vascularly isolated and perfused in situ at a constant flow (6-7 ml/min/100 g). Endotoxemia was induced by a 30 min intravenous infusion of Escherichia coli endotoxin (2 mg/kg), which induced a 50% reduction in arterial pressure. Perfusion pressure, mean arterial pressure, and arteriovenous oxygen difference (a-v O2) were continuously measured. We found 1) no significant difference between the amount of O2 extracted by an innervated or a denervated muscle during endotoxemia; 2) the intra-arterial infusion of norepinephrine or epinephrine into a denervated gracilis muscle (plasma molar concentrations of; 10(-11), 10(-9), 10(-7), and 10(-5) failed to increase MVO2 to the level observed during endotoxemia; 3) pretreatment of a muscle with propranolol to block skeletal muscle beta-adrenergic receptors, did not suppress the endotoxin-induced rise in MVO2. We concluded that the increase in MVO2 seen after the administration of endotoxin is not due to either increased sympathetic nerve activity or elevated levels of circulating catecholamines. We speculate that the increased MVO2 during endotoxemia is caused by nonadrenergic mediators released by endotoxin rather than the hypotensive stimulus.

The goals of this study were to determine 1) if endotoxin alters vascular responsiveness to metabolic stimuli and 2) if the decompensatory loss of skeletal muscle vascular tone that occurs during endotoxemia is induced by increased muscle metabolism. Vascularly isolated and denervated canine gracilis muscles were perfused in situ at a constant flow. In the first set of experiments, gracilis muscle O2 extraction (MVO2) and perfusion pressure were continuously measured during direct electrical stimulation of the muscle mass. Endotoxemia was induced by a 30-min intravenous infusion of Escherichia coli endotoxin (2 mg/kg), and the stimulations were repeated 60 min postendotoxemia. Compared with the nonendotoxic control, the endotoxemic muscle stimulation resulted in a decreased MVO2, and the vascular response (dilation) was potentiated. In the second set of experiments, the MVO2 of the experimental muscle (GMe) was lowered by cooling the temperature of the blood perfusing the muscle to 22-24 degrees C while maintaining the temperature of the contralateral control muscle (GMc) at 34-35 degrees C. After the administration of endotoxin, arterial pressure fell and the GMc showed a progressive increase in MVO2 and loss of vascular tone (decompensation). Coincidently, the GMe showed no significant change in MVO2 and did not vasodilate. The major findings of this study are 1) endotoxin induces the vasculature to become more reactive to metabolic vasodilation, and 2) the decompensatory vasodilation typically observed during endotoxemia can be abolished if MVO2 (i.e., metabolism) is kept low by cooling the muscle. The data suggest that endotoxemia increases vascular sensitivity to vasodilatory metabolites, which allows local mechanisms to dominate extrinsic nonneural forces and control vascular tone, thus inducing vasodilation.

The principal aim of these experiments was to evaluate the ability of a skeletal muscle to extract oxygen during endotoxemia, and determine if the decompensatory decrease in skeletal muscle vascular resistance that occurs after exposure to endotoxin is related to muscle oxygen uptake (VO2). A vascularly isolated denervated canine gracilis muscle was perfused in situ at a constant flow (5-7 mL/min/100 g). Endotoxemia was induced by a 30-min intravenous infusion of Escherichia coli endotoxin (2 mg/kg). Perfusion pressure and arteriovenous oxygen difference (a-v O2) were continuously measured, and muscle O2 extraction was calculated (VO2 = flow x a-v O2). These studies found that gracilis muscle oxygen uptake increased from a resting value of 0.30 mL O2/min/100 g to 0.63 mL O2/min/100 g (111 % increase) by 90 min post-endotoxin. The arterial conductance (i.e., arterial dilation) increased 58% during this time. The amount of oxygen the muscle extracts was found to be directly related to the degree of vasodilation (r = .97), and inversely correlated to mean arterial pressure (r = .97). Pre-dilating the muscle with sodium nitroprusside did not alter oxygen extraction. However, after the introduction of endotoxin, a pre-dilated muscle increased VO2 94% by.90 min. These observations support the concept that endotoxin causes an increase in VO2 without producing a defect in the ability of muscle to extract oxygen. The vasodilation typically observed in skeletal muscle during endotoxemia is not the cause of the increased oxygen uptake. It seems likely that mediators released by endotoxin metabolically stimulate skeletal muscle cells, which increases oxygen demand, thus promoting vasodilation.

The primary aim of these studies was to specifically evaluate the non-neural role of the cyclooxygenase products on the peripheral vascular decompensation associated with endotoxemia. The constant-flow perfused, vascularly isolated, denervated double-canine gracilis muscle preparation in which one muscle is used as a control for the contralateral side was employed. The experimental muscle (GMi) received ibuprofen while the control (GMc) was infused with the vehicle. The results of these studies suggest that endotoxin increases the arterial conductance (i.e., arterial dilation) by 100% and venous compliance (i.e., venoconstriction) by 40%. These observations, which are consistent with venous pooling, were not significantly altered by the continuous intra-arterial infusion of ibuprofen at a peripheral blood concentration of 160 microM. Ibuprofen caused a small but statistically significant increase in the conductance/compliance ratio at 60, 75, and 90 min post endotoxin, suggesting that cyclooxygenase products may play a minor role in the non-neural regulation of capillary fluid dynamics during endotoxemia. Consequently, these studies do not provide convincing evidence that would support a non-neural cyclooxygenase role in the peripheral vascular decompensation reported to occur during systemic endotoxemia.

The purpose of this study was to determine if endogenously released histamine and its non-neural interaction with the H1- and H2-histaminergic receptors in the peripheral vasculature can account for the decompensatory loss of peripheral vascular tone associated with the hypotension occurring during endotoxemia. A denervated in situ constant flow double canine gracilis muscle preparation that permitted one muscle to serve as a control (GMc) for the contralateral experimental muscle (GMe) was used. Endotoxemia was induced by intravenous infusion of 2 mg.kg-1.30 min-1 endotoxin. The specific H1 and H2 antagonists diphen-hydramine and cimetidine were infused either together or separately in both high and low dosages into the GMe. Blockades were validated by intra-arterial injection of histamine or the specific agonists betahistine for H1 and dimaprit for H2 receptors. The results suggest that the high-dose diphenhydramine produced a nonspecific dilation not seen with the lower dose. Because both the blocked and unblocked vascular beds exhibited the same degree of vasodilation after endotoxin, these studies do not support the hypothesis that endogenously released histamine is responsible for the loss of vascular tone. These studies do verify, however, that a nonneurally mediated loss of skeletal muscle vascular tone is an important factor to consider in the overall cardiovascular hypotension occurring during endotoxin shock.